Our past studies have led us to conclude that the most significant developmental and age-related changes in the major cartilage proteoglycan, aggrecan, occur within its glycosaminoglycan chains. The two Specific Aims focus on our hypothesis that the glycosaminoglycan chains of proteoglycans contain chemical and structural information which affects/controls cartilage extracellular matrix structure and function. The availability of specific monoclonal antibodies to unique carbohydrate domains provides the tools to analyze these chains of aggrecan with a high resolving power. Two independent but overlapping experimental programs will be conducted. The first is to use anti-chondroitin sulfate monoclonal antibodies in an affinity purification system to separate high affinity and low affinity immunoreactive chains from each other, as well as from non-immunoreactive chondroitin sulfate chains. Subsequently, the chemical and other structural characteristics that differentiate these subpopulations will be defined. Following partial enzymatic depolymerization of chains in each population, gel chromatography will be used to obtain the hexasaccharide component of the digestion products. Immunoreactive hexasaccharides, obtained by immuno-affinity procedures, will be fractionated by high pressure liquid chromatography into different subpopulations based upon their chemical structures. The resulting pools of chemically homogeneous hexasaccharides will then be used for chemical sequencing. Three independent sequence strategies will be used to define the structures of the minimal motifs recognized by each antibody. Antibody 7D4 will be used in the initial studies to set guidelines for characterization of other epitopes recognized by different anti- chondroitin sulfate antibodies. Concurrently, maps of individual immunoreactive domains will be established by assaying oligosaccharide fragments obtained from different regions of chondroitin sulfate for immunoreactivity and structure. The second program will use these antibodies and the chemical and structural information gained from the first experimental program to study subtle changes in chondroitin sulfates of chick aggrecan obtained from chick limb bud chondrocytes cultured and extracted at different developmental stages corresponding to newly differentiating, mature, and aging chondrocytes. We also propose to use this technology on in vivo cartilage specimens from different anatomic sites and at the same site of different aged individuals to validate our in vitro data. The detailed chemical analyses obtained from the first part of the program will thus be applied to understanding how the chemistry of chondroitin sulfate is modified during development and aging in culture and in vivo. The refinement and further development of this technology will allow us to more precisely comprehend the chemical and molecular basis for changes in the mechanical and functional properties of cartilage during the normal process of development and aging as well as during pathologic changes in human and other vertebrate cartilages.